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Introduction to Microbial Media

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Microbiological studies require the availability of different species of bacteria, yeast or viruses in the laboratory. Large scale processes such as fermentation, protein and vaccine production require large numbers of bacteria in physiologically active state. A suitable nutrient medium that provides appropriate biochemical environment and maintains all the characteristics of microbes is therefore required for various applications.

Any microbial medium should include sources of nutrition, energy, essential minerals, buffering agents and pH indicators. Sometimes they also include selective agents and solidifying agents. Obligate parasites such as bacteriophages may be cultivated in a nutrient medium that also contains host bacteria.

The following are the essential components of a typical microbial medium:

  • Nutrition sources – sources of proteins, vitamins, mineral and carbohydrates; mostly mixture of peptone and meat extract
  • Energy sources – sources of carbohydrates; mostly glucose
  • Essential minerals – sources of micro and macro minerals; mostly present in peptone and meat extract
  • Buffering agents – agents that maintain optimum pH; mostly specific amino acids, phosphates, citrates and zwitterions
  • pH indicators – agents that indicate variation in pH of the medium by change in color; mostly phenol red
  •  Selective agents – agents that allow the growth of only specific bacteria; antibiotics, tellurites, azides and bile salts are examples
  • Solidifying agents – agents that maintain the medium in solid or semi-solid state, if required; widely used agent is agar though gelatin is sometimes used
  • Specific substances such as growth factors, enzymes may be incorporated into the medium for specific bacteria

Types of microbial media

  • Simple or basal media: Include nutrient broth and peptone water; used routinely to isolate and culture a variety of bacteria in a molecular biology research laboratory
  • Complex media: Contain mixture of a variety of nutrients; the exact composition of amino acid source is not defined. Examples include chocolate agar, MacConkey agar, Lowenstein-Jensen medium
  • Defined media: Contain known and defined sources of carbon and nitrogen as required by certain bacteria
  • Special media: Contain specific substances that encourage growth of fastidious bacteria, discourage the growth of unwanted bacteria or contain indicators that distinguish different types of bacteria

Sigma’s Microbial Media

Sigma-Aldrich offers a range of Microbial media for the culture of bacteria, yeast and bacteriophages.

LB medium: Lysogeny Broth or LB is the most popular and commonly used medium to culture E. coli. It was first created by Bertani G to culture Shigella indicator strain. LB is a rich medium containing peptone, yeast extract, NaCl and agar (for solid medium). Depending on the concentration of NaCl, the LB medium may be classified as follows:

LB – Miller: contains 10 g/L peptone, 5 g/L yeast extract, 10 g/L NaCl (and 15 g/L agar for solid medium); suitable for regular E. coli culture

LB – Lennox: contains 10 g/L peptone, 5 g/L yeast extract, 5 g/L NaCl (and 15 g/L agar for solid medium); suitable for culture of E. coli strains that are salt-sensitive

LB – low salt: contains 10 g/L peptone, 5 g/L yeast extract, 0.5 g/L NaCl (and 15 g/L agar for solid medium); suitable for culture of E. coli strains that require low salt conditions to grow

  • LB media are suitable for non-selective cultivation of E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added. Plates containing sterile LB agar with or without antibiotics are also available for selective culture of antibiotic-resistant strains of E. coli.

Minimal salts (M9) medium:  M9 medium contains essential salts and nitrogen. It contains 33.9 g/L Na2HPO4.7H2O, 15 g/L KH2PO4, 5 g/L NH4Cl and 2.5 g/L NaCl; suitable for recombinant E. coli strains

  • Minimal salts (M9) medium is suitable for non-selective cultivation of E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.

Terrific Broth: Terrific broth is a nutrient rich medium that results in higher yield of bacteria in less time. It contains 23.6 g/L yeast extract, 11.8 g/L tryptone, 9.4 g/L K2HPO4 and 2.2 g/L KH2PO4.

  • Terrific broth (modified) is suitable for non-selective cultivation of E. coli strains for cloning and high yield production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.

EnPresso™ B Growth Systems: EnPresso™ B Growth Systems are proprietary formulations of buffering salts, amino acids, carbon source and polysaccharides.

  • EnPresso™ B Growth Systems are designed to yield at least 5x more protein compared to traditional culture media.

SOB medium: SOB medium has higher peptone, amino acids and peptides than LB medium. It contains 20 g/L tryptone, 5 g/L yeast extract, 2.4 g/L MgSO4, 0.5 g/L NaCl and 0.186 g/L KCl.

  • Hanahan’s Broth (SOB medium) enables the preparation of high efficiency competent cells.

SOC medium: SOC medium is a modified SOB medium with added glucose. In the presence of glucose the cellular processes utilize other sugars are repressed. The expression of recombinant proteins that might affect the host cell physiology is inhibited thus resulting in high efficiency transformation reactions. SOC medium contains 20 g/L tryptone, 5 g/L yeast extract, 4.8 g/L MgSO4, 3.603 g/L dextrose, 0.5 g/L NaCl and 0.186 g/L KCl.

  • SOC medium enables the high efficiency transformation of competent cells.

2X YT medium: 2X YT medium contains double the concentration of yeast extract compared to typical LB medium. It is useful for the growth and maintenance of high density of cells for a longer growth period. It contains 16 g/L tryptone, 10 g/L yeast extract and 5 g/L NaCl.

  • 2X YT medium is suitable for non-selective cultivation of M13 bacteriophage and E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.

NZCYM Broth: NZCYM Broth is nutrient rich broth with amino acids, peptides, vitamins and low salt concentration. It contains 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 1 g/L Casamino acids and 1 g/L MgSO4.

  • NZCYM Broth is suitable is suitable for non-selective cultivation of lambda bacteriophage and E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins. It is also suitable for selective cultivation when appropriate antibiotics are added.

Yeast Nitrogen Base: Yeast Nitrogen Base is nutrient-rich medium with sources of nitrogen, vitamins, trace elements and salts. It acts as the basic nutrient medium for most fermentation reactions involving yeast cultures.

  • Yeast Nitrogen Base with and without added amino acids for the cultivation of yeast and for classification based on amino acid and carbon requirements, respectively.

NZ Amine® Broth: NZ Amine® Broth is nutrient rich nutrient-rich medium with amino acids, peptides, vitamins and low salt concentration. It contains 10 g/L tryptone, 5 g/L NaCl and 1 g/L MgSO4.

  • NZ Amine® Broth is suitable for the growth of lambda bacteriophage.

Sigma’s Microbial Media – Selection Guide

Product No.             Name     Format   Preparation Notes
Bacterial Media
L2542 LB – Miller Liquid Ready-to-use
L3522 Powder Mix 25 g in 1L water; autoclave and cool before use
L3147 Powder with agar Mix 40 g in 1L water, heat to dissolve; autoclave and cool before use
L5667 Plates Ready-to-use
L0168
L0543
L0418
L8795
L3022
LB – Lennox
Powder Mix 20 g in 1L water; autoclave and cool before use
L2897 Powder with agar Mix 35 g in 1L water, heat to dissolve; autoclave and cool before use
L7275 Tablets Add each 1.2 g tablet to 48.3 mL water; autoclave and cool before use
L7025 Tablets with agar Add each 1.68 g tablet to 48.3 mL water; autoclave and cool before use
L7658 EZMix™ Mix 20.6 g in 1L water; autoclave and cool before use
L7533 EZMix™ with agar Mix 35.6 g in 1L water, heat to dissolve; autoclave and cool before use
L5542 Plates Ready-to-use
L9234 Liquid Ready-to-use
L3397
LB – low salt Powder Mix 15.5 g in 1L water; autoclave and cool before use
L3272 Powder with agar
Mix 30.5 g in 1L water, heat to dissolve; autoclave and cool before use
L9233 Liquid Ready-to-use
M6030 Minimal salts (M9)
Powder Mix 56.4 g in 1L water to obtain 5X stock solution; autoclave, cool and dilute to 1X before use
M9956 Liquid Ready-to-use
T5574 Terrific Broth - modified Liquid Ready-to-use
T9179 EZMix™ Mix 48.2 g in 1L water, add 4 mL glycerol; autoclave and cool before use
B11001
EnPresso B Tablets Add tablet to water and shake to dissolve
B11101 EnPresso B Animal-free Tablets Add tablet to water and shake to dissolve
B21201 EnPresso B Defined Nitrogen-free Tablets Add tablet to water and shake to dissolve
H8032 Hanahan’s Broth (SOB Medium)
Powder Mix 28 g in 1L water; autoclave and cool before use
H9950 Liquid Ready-to-use
S1797 SOC Medium Liquid Ready-to-use
Y1003 2X YT medium Liquid Ready-to-use
Y2377 Powder Mix 31 g in 1L water; autoclave and cool before use
Y2627 EZMix™ Mix 31.6 g in 1L water; autoclave and cool before use
N3643 NZCYM Broth Powder Mix 22 g in 1L water; autoclave and cool before use
N6905 EZMix™ Mix 22.6 g in 1L water; autoclave and cool before use
N9920 Liquid Ready-to-use
Yeast Media
Y1250 Yeast Nitrogen Base with amino acids Powder Mix 6.7 g in 100 mL water, add 5 g glucose to obtain 10X stock solution; filter sterilize and dilute to 1X with sterile water before use
Y0626 Yeast Nitrogen Base without amino acids Mix 6.8 g in 100 mL water, add 5 g glucose and 5-10 mg of appropriate amino acids to obtain 10X stock solution; filter sterilize and dilute to 1X with sterile water before use
Phage Media
N3518 NZ Amine®Broth Powder Mix 16 g in 1L water; autoclave and cool before use

 

 References